The present invention generally provides devices, compositions of matter, kits, systems and methods for detecting and identifying a plurality of signals from within a signal area. In a particular embodiment, the invention provides systems and methods for detecting and identifying a plurality of spectral barcodes from throughout a sensing area, especially for identifying and/or tracking inventories of elements, for high-throughput assay systems, and the like. The invention will often use labels which emit identifiable spectra that include a number of discreet signals having measurable wavelengths and/or intensities.
Tracking the locations and/or identities of a large number of items can be challenging in many settings. Barcode technology in general, and the Universal Product Code in particular, has provided huge benefits for tracking a variety of objects. Barcode technologies often use a linear array of elements printed either directly on an object or on labels which may be affixed to the object. These barcode elements often comprise bars and spaces, with the bars having varying widths to represent strings of binary ones, and the spaces between the bars having varying widths to represent strings of binary zeros.
Barcodes can be detected optically using devices such as scanning laser beams or handheld wands. Similar barcode schemes can be implemented in magnetic media. The scanning systems often electro-optically decode the label to determine multiple alphanumerical characters that are intended to be descriptive of (or otherwise identify) the article or its character. These barcodes are often presented in digital form as an input to a data processing system, for example, for use in point-of-sale processing, inventory control, and the like.
Barcode techniques such as the Universal Product Code have gained wide acceptance, and a variety of higher density alternatives have been proposed. Unfortunately, these standard barcodes are often unsuitable for labeling many xe2x80x9clibrariesxe2x80x9d or groupings of elements. For example, small items such as jewelry or minute electrical components may lack sufficient surface area for convenient attachment of the barcode. Similarly, emerging technologies such as combinatorial chemistry, genomics research, microfluidics, micromachines, and other nanoscale technologies do not appear well-suited for supporting known, relatively large-scale barcode labels. In these and other developing fields, it is often desirable to make use of large numbers of fluids, and identifying and tracking the movements of such fluids using existing barcodes is particularly problematic. While a few chemical encoding systems for chemicals and fluids have been proposed, reliable and accurate labeling of large numbers of small and/or fluid elements remains a challenge.
Small scale and fluid labeling capabilities have recently advanced radically with the suggested application of semiconductor nanocrystals (also known as Quantum Dot(trademark) particles), as detailed in U.S. patent application Ser. No. 09/397,432, the full disclosure of which is incorporated herein by reference. Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. As the band gap energy of such semiconductor nanocrystals vary with a size, coating and/or material of the crystal, populations of these crystals can be produced having a variety of spectral emission characteristics. Furthermore, the intensity of the emission of a particular wavelength can be varied, thereby enabling the use of a variety of encoding schemes. A spectral label defined by a combination of semiconductor nanocrystals having differing emission signals can be identified from the characteristics of the spectrum emitted by the label when the semiconductor nanocrystals are energized.
While semiconductor nanocrystal-based spectral labeling schemes represent a significant advancement for tracking and identifying many elements of interest, still further improvements would be desirable. In general, it would be beneficial to provide improved techniques for sensing or reading these new spectral labels. It would be particularly beneficial to provide improved techniques for applying these labeling and tracking technologies to high-throughput assay systems now being developed.
Multiplexed assay formats would be highly desirable for improved throughput capability, and to match the demands that combinatorial chemistry is putting on established discovery and validation systems for pharmaceuticals. For example, simultaneous elucidation of complex protein patterns may allow detection of rare events or conditions, such as cancer. In addition, the ever-expanding repertoire of genomic information would benefit from very efficient, parallel and inexpensive assay formats. Desirable multiplexed assay characteristics include ease of use, reliability of results, a high-throughput format, and extremely fast and inexpensive assay development and execution.
A number of known assay formats may be employed for high-throughput testing. Each of these formats has limitations, however. By far the most dominant high-throughput technique is based on the separation of different assays into different regions of space. The 96-well plate format is the workhorse in this arena.
In 96-well plate assays, the individual wells (which are isolated from each other by walls) are often charged with different components, and the assay is performed and then the assay result in each well measured. The information about which assay is being run is carried with the well number, or the position on the plate, and the result at the given position determines which assays are positive. These assays can be based on chemiluminescence, scintillation, fluorescence, scattering, or absorbance/colorimetric measurements, and the details of the detection scheme depend on the reaction being assayed.
Multi-well assays have been reduced in size to enhance throughput, for example, to accommodate 384 or 1536 wells per plate. Unfortunately, the fluid delivery and evaporation of the assay solution at this scale are significantly more confounding to the assays. High-throughput formats based on multi-well arraying often rely on complex robotics and fluid dispensing systems to function optimally. The dispensing of the appropriate solutions to the appropriate bins on the plate poses a challenge from both an efficiency and a contamination standpoint, and pains must be taken to optimize the fluidics for both properties. Furthermore, the throughput is ultimately limited by the number of wells that one can put adjacent on a plate, and the volume of each well. Arbitrarily small wells have arbitrarily small volumes, resulting in a signal that scales with the volume, shrinking proportionally with the cube of the radius. The spatial isolation of each well, and thereby each assay, has been much more common than running multiple assays in a single well. Such single-well multiplexing techniques are not widely used, due in large part to the difficulty in xe2x80x9cdemultiplexingxe2x80x9d or resolving the results of the different assays in a single well.
For even higher throughput genomic and genetic analysis techniques, positional array technology has been shrunk to microscopic scales, often using high-density oligonucleotide arrays. Over a 1-cm square of glass, tens to hundreds of thousands of different nucleotides can be written in, for example, 25-xcexcm spots, which are well resolved from each other. On this planar test structure or xe2x80x9cchip,xe2x80x9d which is emblazoned with an alignment grid, a particular spot""s x,y position determines which oligonucleotide is present at that spot. Typically fluorescently-labeled amplified DNA is added to the array, hybridized and is then detected using fluorescence-based techniques. Although this is a very powerful technique for assaying a large number of genetic markers simultaneously, the cost is still very high, and the flexibility of this assay is extremely limited.
Once a chip is made with particular DNA sequences at particular locations, they are fixed and the addition thereto of new markers comes at a very high price. The extremely small feature size, and the highly parallel assay format, comes at the cost of the flexibility inherent in a common platform system, such as the 96-well plates. In addition, this assay is ultimately performed at the surface of the chip, and the results depend on the kinetics of the hybridization to the surface, a process that is negatively influenced by steric issues, mixing issues, and diffusion issues. In fact, small microarray chips are not particularly suited to the detection of rare events, as the diffusion of the solution over the chip may not be sufficiently thorough. In order to perform the hybridizations to the microarray chips more efficiently, a dedicated fluidics workstation can be used to pump the solution over the surface of the chip repeatedly; such instruments add cost and time to execution of the assay.
The use of spectral barcodes holds great promise for enhancing the throughput of assays, as described in an application entitled xe2x80x9cSemiconductor Nanocrystal Probes for Biological Applications and Process for Making and Using such Probes,xe2x80x9d U.S. Application Ser. No. 09/259,982 filed Mar. 1, 1999, the full disclosure of which is incorporated herein by reference. Multiplexed assays may be performed using a number of probes which include both a spectral label (often in the form of several semiconductor nanocrystals) and one or more moieties. The moieties may be capable of selectively bonding to one or more detectable substances within a sample fluid, while the spectral labels can be used to identify the probe within the fluid (and hence the associated moiety). As the individual probes can be quite small, and as the number of barcodes which can be independently identified can be quite large, large numbers of individual assays might be performed within a single fluid sample by including a large number of differing probes. These probes may take the form of quite small beads, with each bead optionally including a spectral label, a moiety, and a bead body or matrix, often in the form of a polymer.
Together with the substantial advantages provided by highly multiplexed, spectrally-encoded assay bead systems, there will be significant challenges in implementing these techniques. In particular, determining multiplexed assay results might be quite challenging. While the reaction times and accuracy of the spectral labels can be quite advantageous, it can be challenging to accurately read each spectral barcode and/or assay result from the hundreds, and in many cases thousands, of beads within a highly multiplexed bead assay system. Similarly, while spectral coding in general allows labeling and/or identification of a large number of elements, interpreting the spectral codes can be quite challenging when the individual label structures are small, and when many labels are located near each other.
In light of the above, it would generally be desirable to provide improved systems and methods for detecting and identifying signals. It would be particularly beneficial if these improved techniques facilitated the identification of each spectral code from among a plurality of spectral barcodes in a given region. To take advantage of the potential capabilities of spectral coding of minute probes and other structures, it would be highly desirable if these enhanced techniques allowed detection and/or identification of large numbers of spectral codes or other signals (such as assay marker signals) in a highly time efficient manner.
The present invention generally provides improved devices, systems, and methods for sensing and/or identifying signals. The techniques of the present invention are particularly well-suited for identification of labels which generate spectral codes. Large numbers of independently identifiable spectral codes can be generated by quite small bodies having such labels, and a plurality of such bodies or probes may be present within a detection region. In some embodiments, the invention allows simultaneously imaging of identifiable spectra from throughout the detection region. This simultaneous imaging allows the labels (and hence, the associated probes, assay results, and the like) to be identified. A wavelength dispersive element (for example, a prism, diffractive grating, holographic transmissive grating, or the like) can simultaneously spectrally disperse the images of the labels across a sensor surface. A two-dimensional areal light sensor (such as a Charge-Coupled Device or xe2x80x9cCCDxe2x80x9d) can substantially simultaneously sense the relative wavelengths of signals making up the spectra. Taking advantage of a very small label size, the identifiable spectra can be treated as being generated from point-sources within a large detection field, thereby acting as their own xe2x80x9cslitxe2x80x9d in this spectroscopic instrument. Absolute signal wavelengths may be identified by determining positions of the labels, using an internal wavelength reference within the spectra, and/or the like.
Spectral labels may be used with other markers generating signals that differ significantly from the identifiable spectra from the labels. For example, spectrally encoded beads may be used within parallel assay systems by generating assay signals in addition to the label spectra. These assay signals may accurately and reliably indicate the results of the assay, but these signals may be significantly lower in intensity than the spectral label. Hence, the present invention also provides techniques for identifying signals of widely varying strengths. These techniques often involve simultaneously sensing lower intensity signals using a relatively long integration time with areal imaging. Higher intensity signals can be sequentially sensed, often using a scanning system. This dual sensing system enhances the overall efficiency of signal detection and interpretation by allowing a relatively long signal integration time for the lower intensity signals, while the higher intensity signals are quickly scanned with a shorter integration time. In some embodiments, a plurality of excitation energies may be directed toward the signal generators, with at least one of the excitation energies selectively producing the lower energy signals. Such techniques are particularly well-suited to take advantage of the capabilities of semiconductor nanocrystals, which can accurately generate detectable signals from minute bodies, and which can be selectively energized by appropriate excitation sources.
In a first aspect, the invention provides a system comprising a plurality of labels generating identifiable spectra in response to excitation energy. A detector simultaneously images at least some of the spectra for identification of the labels.
In many embodiments, at least some of the spectra will comprise a plurality of detectable signals defining a plurality of wavelengths. Label markers may generate these different label signals, so that the labels can comprise a plurality of label markers. The wavelengths from the spectra can be intermingled. Preferably, the labels will comprise at least one semiconductor nanocrystal. More typically, each label will comprise at least one population of semiconductor nanocrystals, with each semiconductor nanocrystal of each population generating a signal having an associated population wavelength in response to the excitation energy. In many embodiments, the labels will comprise a plurality of populations supported by a matrix.
In some embodiments, at least one probe body will include a label and an associated assay indicator marker. The indicator markers generate indicator signals in response to an interaction between the probe body and an associated test substance, thereby indicating results of an assay.
The labels may be distributed across a two-dimensional sensing field. The detector will often include a wavelength dispersive element and a sensor, and each label will preferably be sufficiently smaller than the surrounding sensing field to allow the spectra to be wavelength-dispersed by the wavelength dispersive element without excessive overlap of the dispersed spectra upon the sensor. The dispersed spectra can often be analyzed as being generated from discrete point-light sources. By using discrete point source spectral labels, the system avoids any need for slit apertures or the like, as generally found on linear spectrometers and other spectral dispersion systems. In other words, the small labels can act as their own slits. This also allows the detector to admit signals from throughout a two-dimensional sensing field.
The wavelength dispersive element is usually disposed between the sensing field and the light sensor. The sensor simultaneously senses the spectra from the plurality of labels. An open optical path often extends from the sensing field to the wavelength dispersive element, and from the wavelength dispersive element to the sensor, with optics typically imaging the sensing field on the sensor. The sensor will typically comprise an areal sensor (such as CCD), and the open optical path will have an open cross-section with significant first and second open orthogonal dimensions, in contrast to the slit or point apertures often used in dispersive systems. The wavelength dispersive element may comprise a prism, a dispersive reflective grating, a holographic transmission grating, or the like.
In many embodiments, a spatial positioner provides label positions within the sensor field. The detector will often sense relative spectral data, while an analyzer coupled to the label positioner and the detector can derive absolute wavelengths of the spectra in response to both the relative spectral data and the indicated label positions. In some embodiments, a beam splitter may optically couple the label positioner with the sensing field along a positioning optical path, and may also couple the detector with the sensor field along a spectral optical path, so that at least a portion of the positioning and spectral optical paths make use of common optical elements. The beam splitter may direct most of the energy from the sensing field toward the detector for relative spectral information, and a minority of the energy from the sensing field toward a positioning image. In some embodiments, a beam splitter may direct a portion of an image from the sensing field to a first dispersion member so as to distribute the spectra along a first axis relative to the sensing field, and a second portion of the image to a second dispersion member so as to distribute the spectra along a second axis, the second axis being at an angle to the first axis relative to the sensing field for resolving spectral ambiguities from any overlapping wavelengths along the first axis. Similar ambiguity resolution techniques may sequentially disperse the spectra along differing axes.
At least some of the spectra will often comprise a plurality of signals. The detector may include means for distributing these signals across a sensor in response to wavelengths of the signals, and in response to positions of the labels in the sensor fields. The distributing means may be disposed between the sensing field and the sensor. The system may also include means for determining positions of the labels within the sensing field, with a spectral analyzer coupled to the positioning means and the sensor so that the analyzer can determine the spectra. The positioning means may optionally comprise an areal sensor and a beam splitter, a calibration reference signal within some or all of the spectra, or the like.
In another aspect, the invention provides a system comprising a plurality of labels distributed across a two-dimensional sensing field. The labels generate spectra in response to excitation energy. A wavelength dispersive element is disposed in an open optical path of the spectra from the two-dimensional sensing field. A sensor is disposed in the path from the wavelength dispersive element. A label positioning system is coupled to the labels and an analyzer is coupled to the sensor for identifying the labels in response to the sensed spectral information.
In another aspect, the invention provides a method comprising generating spectra from a plurality of labels. The spectra are sensed with a sensor by simultaneously imaging the labels on the sensor, and the labels are identified in response to the sensed spectra.
In many embodiments, the labels will be movably disposed within a two-dimensional sensing field while the spectra are sensed. The positions of the labels may be determined when the spectra are sensed by the sensor, and the labels may be identified in response to the label positions (as well as using the data from the sensor). The spectra from the labels will often be dispersed. In some embodiments, the spectra will be dispersed along a second dispersion axis at an angle to a first dispersion axis so as to resolve ambiguity from spectral overlap.
In another aspect, the invention provides a method for identifying signals of differing strengths. The method comprises generating a plurality of signals in response to excitation energy. The signals include higher intensity signals and lower intensity signals. The lower intensity signals are sensed by simultaneously imaging the signals. At least some of the higher intensity signals are sequentially sensed.
In many embodiments, the lower intensity signals will be sensed by imaging a sensing field for a first integration time. The higher intensity signals may be sequentially sensed by imaging a portion of the sensing field for a second integration time, the second integration time being shorter than the first integration time. Optionally, the higher intensity signals may be filtered from the simultaneous image. This is facilitated where the higher intensity signals have wavelengths that are different than wavelengths of the lower intensity signals, as wavelength filtering may be employed to avoid saturation of the image.
The higher intensity signals may be sequentially sensed by scanning labels which generate the signals. The labels generating the higher intensity signals may be spatially intermingled with markers generating the lower intensity signals. Scanning may comprise scanning an aperture relative to the labels, such as a slit, a pinhole aperture, or the like. In some embodiments, scanning may be performed by scanning an excitation energy over a portion of the sensing field.
In some embodiments, the excitation energy may comprise a first energy for exciting the higher energy markers of the labels to generate the high energy signals, and a second energy for generating the lower energy signals. The second energy may selectively excite the low energy markers.
The higher intensity signals of the labels may be generated by label markers and can define an identifiable spectral code. The low intensity signals may be generated by assay markers and can indicate results of a plurality of assays, with each assay having an associated spectral code. The markers may be supported by probe bodies to define probes. Each probe can include a plurality of label markers, which together define a label (to generate the spectral code), and at least one associated assay marker (to indicate results of an associated assay). The results of each assay may be determined by identifying each label, and by correlating the label with an associated assay marker signal.
In another aspect, the invention provides a method for acquiring signals. The method comprises generating a first plurality of signals from a first plurality of markers in response to a first excitation energy. A second plurality of signals are generated from a second plurality of markers in response to a second excitation energy. The first and second markers are intermingled. Intensities of the first signals are tuned relative to intensities of the second signals by selecting a characteristic of at least one of the first and second excitation energies. The tuned first and second signals are simultaneously imaged on a sensor.
Typically, at least one of the markers will comprise a semiconductor nanocrystal. Preferably, the first energy will selectively energize the first plurality of markers. The intensities will be tuned so that the signals are within an acceptable intensity range of the sensor during a common integration time by varying an intensity of at least one of the first and second excitation energies.
In yet another aspect, the invention provides a high-throughput assay method comprising performing a plurality of assays, and generating assay signals with assay markers to indicate the results of the assays. The assay markers are simultaneously area imaged, and spectral codes associated with each assay marker are generated. The assay results are interpreted by identifying the spectral code and assay markers, and by correlating each spectral code with an associated assay marker signal.
In another aspect, the invention provides a system for detecting spectral information. Spectral information includes higher intensity signals and lower intensity signals. The signals are generated within a two-dimensional field. The systems comprises a detector optically couplable with the two-dimensional field for simultaneous imaging of the low intensity signals. A scanner has an aperture movable relative to the two-dimensional field for sequential imaging of the higher intensity signals.
In yet another aspect, the invention provides a system comprising a plurality of labels generating identifiable spectra in response to excitation energy. Other markers are intermingled with the labels. The other markers generate other signals, with the other signals being weaker than the spectra. A scanner has an aperture movable relative to the labels for identifying the spectra. A detector is optically coupled to the plurality of other markers for simultaneously imaging the other signals.
Typically, groups of the markers will be held together by a probe matrix so as to define a plurality of probes, with each probe including at least one label and at least one associated other marker. This allows each probe to indicate results of an associated assay via the identifiable spectra of the label. A processor coupled to the scanner and to the detector can determine the results of the assay in response to the spectra as sensed by the scanner, and in response to the associated assay markers as sensed by the detector. An integration time of the detector can be longer than an integration time of the scanner for the spectra without overly delaying the identification time, as the other markers (or assay markers) are simultaneously imaged throughout the sensing field.
In yet another aspect, the invention provides a high-throughput assay system comprising a fluid with an excitation energy source transmitting excitation energy toward the fluid. A plurality of assay probes are disposed in the fluid. Each probe has a spectral label. The spectral labels generate identifiable spectral codes in response to the excitation energy. The probes generate assay signals in response to assay results. A scanner moves a sensing region relative to the fluid (and/or at least one of the fluid and fluid holder relative to the sensing region) for identification of the probes from the spectral codes. The two-dimensional imaging system images the assay markers from the probes throughout the two-dimensional sensing field simultaneously.
In yet another aspect, the invention provides a high-throughput assay system comprising a fluid and a first excitation energy source transmitting a first excitation energy toward the fluid. The second excitation energy source transmits a second excitation energy toward the fluid. A plurality of assay probes are disposed in the fluid. Each probe has a spectral label, and assay markers in the fluid are associated with the probes. The assay markers transmit an assay signal in response to assay results, and in response to the second excitation energy. A first excitation energy selectively energizes the spectral labels so that the spectral labels transmit identifiable spectral codes. A sensing system senses the assay signals and the spectral codes. The sensing system has an intensity range. Intensities of the first and second excitation sources are selected so that the assay signals and the spectral codes are within the intensity range, often at the same integration time.
In yet another aspect, the invention provides a fluid-flow assay system comprising a fluid and a probe movably disposed within the fluid. The probe has a label to generate an identifiable spectra and an assay marker to generate an assay signal in response to interaction between the probe and a detectable substance. A probe reader senses the spectra and signal when the probe and fluid flow through a sensing region to determine an assay result.
Typically, a plurality of differing probes will flow through the sensing region. The probe reader will determine results of a plurality of different assays by identifying the probes from their associated spectra, and by correlating the assay signals from the probes with the associated assays of the identified probes. In the exemplary embodiment, the fluid (and the probes) flow across a slit aperture within a thin, flat channel so that the distance between the probes and reader is substantially uniform. This facilitates imaging of the probes within the sensing region.
In yet another aspect, the invention provides a fluid-flow assay method comprising moving a probe by flowing a fluid. A spectra from the moving probe is sensed while the probe acts as its own aperture by dispersing the image, and results of an assay are determined by identifying the probe from the spectra. Once again, such methods are particularly useful for multiplexed assays, as a plurality of differing probes can be identified and their assay results correlated.